Meera Iyer

Ex vivo cell labeling with 64Cu–pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography

We have used copper-64-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu–PTSM) to radiolabel cells ex vivo for in vivo positron-emission tomography (PET) imaging studies of cell trafficking in mice and for eventual application in patients. 2-[18F]-Fluoro-2-deoxy-d-glucose (FDG) cell labeling also was evaluated for comparison. 64Cu–PTSM uptake by C6 rat glioma (C6) cells increased for 180 min and then stabilized. The labeling efficiency was directly proportional to 64Cu–PTSM concentration and influenced negatively by serum. Label uptake per cell was greater with 64Cu–PTSM than with FDG. However, both 64Cu–PTSM- and FDG-labeled cells showed efflux of cell activity into supernatant. The 64Cu–PTSM labeling procedure did not interfere significantly with C6 cell viability and proliferation rate. MicroPET images of living mice indicate that tail-vein-injected labeled C6 cells traffic to the lungs and liver. In addition, transient splenic accumulation of radioactivity was clearly detectable in a mouse scanned at 3.33 h postinfusion of 64Cu–PTSM-labeled lymphocytes. In contrast, the liver was the principal organ of tracer localization after tail-vein administration of 64Cu–PTSM alone. These results indicate that in vivo imaging of cell trafficking is possible with 64Cu–PTSM-labeled cells. Given the longer t1/2 of 64Cu (12.7 h) relative to 18F (110 min), longer cell-tracking periods (up to 24–36 h) should be possible now with PET.

Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters

We are developing assays to image tissue-specific reporter gene expression in living mice by using optical methods and positron emission tomography. Approaches for imaging reporter gene expression depend on robust levels of mRNA and reporter protein. Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak relative to stronger but constitutively expressing viral promoters. In this study, we have validated methods to enhance the transcriptional activity of the prostate-specific antigen promoter for imaging by using a two-step transcriptional amplification (TSTA) system. We used the TSTA system to amplify expression of firefly luciferase (fl) and mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk) in a prostate cancer cell line (LNCaP). We demonstrate ≈50-fold (fl) and ≈12-fold (HSV1-sr39tk) enhancement by using the two-step approach. The TSTA system is observed to retain tissue selectivity. A cooled charge-coupled device optical imaging system was used to visualize the amplified fl expression in living mice implanted with LNCaP cells transfected ex vivo. These imaging experiments reveal a ≈5-fold gain in imaging signal by using the TSTA system over the one-step system. The TSTA approach will be a valuable and generalizable tool to amplify and noninvasively image reporter gene expression in living animals by using tissue-specific promoters. The approaches validated should have important implications for study of gene therapy vectors, cell trafficking, transgenic models, as well as studying development of eukaryotic organisms.

Noninvasive quantitative imaging of protein–protein interactions in living subjects

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein—protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-κB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-κB promoter through tumor necrosis factor α. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein–protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein–protein interactions in living subjects. The approaches validated should have important implications for the study of protein–protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein–protein interactions.

Seeing is believing: non-invasive, quantitative and repetitive imaging of reporter gene expression in living animals, using positron emission tomography.

The ability to monitor reporter gene expression in living animals and in patients will permit longitudinal examinations both of somatically transferred DNA in experimental animals and patients and of transgenic constructs expressed in experimental animals. If investigators can non‐invasively monitor the organ and tissue specificity, the magnitude and the duration of gene expression from somatically transferred DNA and from transgenes, conceptually new experimental paradigms will be possible. If clinicians can non‐invasively monitor the location, extent and duration of somatically transferred genes, they will be better able to determine the correlations between expression of therapeutic genes and clinical outcomes. We have developed two reporter gene systems for in vivo reporter gene imaging in which the protein products of the reporter genes sequester positron‐emitting reporter probes. The “PET reporter gene” dependent sequestration of the “PET reporter probes” is subsequently measured in living animals by Positron Emission Tomography (PET). We describe here the principles of PET reporter gene/PET reporter probe in vivo imaging, the development of two imaging systems, and the validation of their ability to non‐invasively, quantitatively and repetitively image reporter gene expression in murine viral gene transfer and transgenic models. J. Neurosci. Res. 59:699–705, 2000

Optical imaging of transferrin targeted PEI/DNA complexes in living subjects

Noninvasive optical bioluminescence imaging systems are important tools for evaluating gene expression in vivo for study of individual and temporal variation in a living animal. In this report, we demonstrate that expression of the firefly luciferase reporter gene (fl) delivered by transferrin (Tf) targeted polyethylenimine (PEI) complexes with, or without, poly(ethylene glycol) (PEG) modifications can be imaged in living A/J mice bearing N2A tumors using a cooled charged coupled device (CCD) camera. Tf–PEI–PEG, Tf–PEI, and PEI (positive control) complexes were tail-vein injected and mice were imaged at 5, 24, 48, and 72 h after complex injection. After imaging, the organs were analyzed ex vivo for firefly luciferase protein (FL) activity. The Tf and PEG modified formulations show significantly (P<0.05) higher FL activity in vivo and ex vivo at the tumor as compared to other organs, including the lungs (a site of high expression with PEI, the positive control). Furthermore, the in vivo bioluminescent signal correlated well (R2=0.83) with ex vivo FL activity. These data support that noninvasive imaging of fl reporter expression can be used to monitor the specificity of Tf–PEI and Tf–PEI–PEG polyplex targeting of N2A tumors in A/J mice.

Applications of molecular imaging in cancer gene therapy.

Gene-based therapy is a promising and flexible therapeutic approach to manage diverse types of cancer. The lack of convincing therapeutic success of current gene therapy protocols in part, can be attributed to the inability to monitor gene expression at the targeted site in the living subject. Linking molecular imaging to gene therapy will enable real-time assessment of the therapeutic process and the refinement of treatment protocols. This review will cover two common imaging modalities, positron emission tomography (PET) and bioluminescence imaging (BLI), used in pre-clinical and clinical gene therapy applications. Strategies to develop more specific and robust cancer gene therapy and imaging approaches will be discussed. Coupling PET to gene therapy of cancer has already been implemented in several clinical studies. This approach would help to improve the efficacy and safety of future gene therapy clinical trials.

Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy.

Non-invasive assessment of transgenic animals using bioluminescence imaging offers a rapid means of evaluating disease progression in animal models of disease. One of the challenges in the field is to develop models with robust expression to image repetitively live intact animals through solid tissues. The prostate-specific antigen (PSA) promoter is an attractive model for studying gene regulation due to its hormonal response and tissue-specificity permitting us to measure signaling events that occur within the native tissues. The use of the GAL4-VP16 activator offers a powerful means to augment gene expression levels driven by a weak promoter. We have used a two-step transcriptional amplification (TSTA) system to develop a transgenic mouse model to investigate the tissue-specificity and developmental regulation of firefly luciferase (fl) gene expression in living mice using bioluminescence imaging. We employed an enhanced prostate-specific promoter to drive the yeast transcriptional activator, GAL4-VP16 (effector). The reporter construct carries five Gal4 binding sites upstream of the fl gene. We generated a transgenic mouse model using a single vector carrying the effector and reporter constructs. The transgenic mice show prostate-specific expression as early as three weeks of age. The bioluminescence signal in the prostate is significantly higher than in other organs. We also demonstrate that blocking androgen availability can downregulate the fl expression in the prostate. The transgenic mice display normal physical characteristics and developmental behavior, indicating that the high level of GAL4 driven expression is well tolerated. These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model. The transgenic TSTA model in conjunction with other transgenic cancer models should also help to detect and track malignancies. The strategies developed will be useful for transgenic research in general by allowing for amplified tissue specific gene expression.

Novel bidirectional vector strategy for amplification of therapeutic and reporter gene expression

Molecular imaging methods have previously been employed to image tissue-specific reporter gene expression by a two-step transcriptional amplification (TSTA) strategy. We have now developed a new bidirectional vector system, based on the TSTA strategy, that can simultaneously amplify expression for both a target gene and a reporter gene, using a relatively weak promoter. We used the synthetic Renilla luciferase (hrl) and firefly luciferase (fl) reporter genes to validate the system in cell cultures and in living mice. When mammalian cells were transiently cotransfected with the GAL4-responsive bidirectional reporter vector and various doses of the activator plasmid encoding the GAL4-VP16 fusion protein, pSV40-GAL4-VP16, a high correlation (r(2) = 0.95) was observed between the expression levels of both reporter genes. Good correlations (r(2) = 0.82 and 0.66, respectively) were also observed in vivo when the transiently transfected cells were implanted subcutaneously in mice or when the two plasmids were delivered by hydrodynamic injection and imaged. This work establishes a novel bidirectional vector approach utilizing the TSTA strategy for both target and reporter gene amplification. This validated approach should prove useful for the development of novel gene therapy vectors, as well as for transgenic models, allowing noninvasive imaging for indirect monitoring and amplification of target gene expression.

Imaging androgen receptor function during flutamide treatment in the LAPC9 xenograft model

The current understanding of the response of androgen receptor to pharmacologic inhibitors in prostate cancer is derived primarily from serum prostate-specific antigen (PSA) levels. In this study, we test whether a novel androgen receptor–specific molecular imaging system is able to detect the action of the antiandrogen flutamide on androgen receptor function in xenograft models of prostate cancer. Adenoviruses bearing an optical imaging cassette containing an androgen receptor–responsive two-step transcriptional amplification system were injected into androgen-dependent and hormone-refractory tumors of animals undergoing systemic time-controlled release of the antiandrogen flutamide. Imaging of tumors with a cooled charge-coupled device camera revealed that the response of AdTSTA to flutamide is more sensitive and robust than serum PSA measurements. Flutamide inhibits the androgen signaling pathway in androgen-dependent but not refractory tumors. Analysis of androgen receptor and RNA polymerase II binding to the endogenous PSA gene by chromatin immunoprecipitation revealed that flutamide treatment and androgen withdrawal have different molecular mechanisms. The application of imaging technology to study animal models of cancer provides mechanistic insight into antiandrogen targeting of androgen receptor during disease progression.

Imaging Mitogen-Activated Protein Kinase Function in Xenograft Models of Prostate Cancer

Mitogen-activated protein kinases (MAPK) play important roles in malignancy. The ability to detect and quantitate MAPKs in live animal models of cancer will facilitate an understanding of disease progression. We have developed a gene expression-based imaging system that detects and quantifies MAPK activity in prostate cancer tumors implanted into severe combined immunodeficient mice. The imaging technology uses a modified version of two-step transcriptional amplification (TSTA). The tissue specificity of gene expression is imparted by an enhanced version of the prostate-specific antigen regulatory region that expresses GAL4-ELK1. GAL4-ELK1 confers MAPK specificity by activating a firefly luciferase (FLuc) reporter gene when the Ets-like transcription factor (ELK) 1 activation domain is phosphorylated by MAPK. FLuc activity in live animals was detected using the Xenogen In vivo Imaging System. We validated the TSTA-ELK1 system by analyzing its response to epidermal growth factor treatment in transfected tissue culture cells and in adenovirus (AdTSTA-ELK1)-injected prostate cancer xenograft tumors. We measured MAPK activity in two well-characterized xenograft models, CWR22 and LAPC9. Although no significant differences in MAPK levels were detected between androgen-dependent and androgen-independent xenografts, the CWR22 models display significantly higher levels of AdTSTA-ELK1 activity versus LAPC9. Western blots of tumor extracts showed that the elevated imaging signal in CWR22 xenografts correlated with elevated levels of phosphorylated extracellular signal-regulated kinase 1/2 but not p38 or c-Jun NH(2)-terminal kinase. We conclude that a gene expression-based optical imaging system can accurately detect and quantify MAPK activity in live animals.